It is often desirable to detect specific DNA sequences in a mixture of sequences or in a sample that contains DNA and other ingredients. For instance, detection of specific DNA sequences is often used to determine whether a particular bacterium is present in a biological sample. Many techniques can be used including nicking endonuclease signal amplification (NESA), polymerase chain reaction (PCR), quantitative PCR (qPCR), and DNA sequencing. Most techniques use some form of amplification of the DNA to increase sensitivity coupled to an additional method for the identification itself. For instance, NESA may be coupled to a DNA amplification technique. With qPCR, the amplification step occurs at the same time as the detection step. This gives qPCR both sensitivity and speed.
One method of DNA amplification that has been used with NESA is rolling circle amplification (RCA), also called extended NESA (Li et al., Enzymatic Signal Amplification of Molecular Beacons for Sensitive DNA Detection, Nucleic Acids Res. 2008; 36(6):e36). In RCA, an oligonucleotide is used to prime a DNA synthesis reaction catalyzed by a strand displacing DNA polymerase. The DNA is a small circle that is continuously copied. In the method described by Li et al., an oligonucleotide was used to bind to the target sequence such that the ends of the oligonucleotide were separated only by a nick. DNA ligase was then used to repair the nick. RCA was performed on the circularized DNA followed by the NESA reaction using molecular beacons specific for the now circular oligonucleotide. The NESA reaction is therefore measuring DNA amplification of the added oligonucleotide rather than directly interacting with the target DNA in a sequence specific manner. This assay is therefore a relatively slow, two-step assay and suffers from a built in background due to circularization that can randomly occur with any oligonucleotide of sufficient length, in the presence of DNA ligase.
There remains a need for reliable and sensitive methods to detect specific nucleotide sequences.